Abstract

The negative regulator Cbl functions as a ubiquitin ligase towards activated receptor tyrosine kinases and facilitates their transport to lysosomes. Whether Cbl ubiquitin ligase activity mediates its negative regulatory effects on cytoplasmic tyrosine kinases of the Syk/ZAP-70 family has not been addressed, nor is it known whether these kinases are regulated via ubiquitylation during lymphocyte B-cell receptor engagement. Here we show that B-cell receptor stimulation in Ramos cells induces the ubiquitylation of Syk tyrosine kinase which is inhibited by a dominant-negative mutant of Cbl. Intact tyrosine kinase-binding and RING finger domains of Cbl were found to be essential for Syk ubiquitylation in 293T cells and for in vitro Syk ubiquitylation. These same domains were also essential for Cbl-mediated negative regulation of Syk as measured using an NFAT-luciferase reporter in a lymphoid cell. Association with Cbl did not alter the kinase activity of Syk. Altogether, our results support an essential role for Cbl ubiquitin ligase activity in the negative regulation of Syk, and establish that ubiquitylation provides a mechanism of Cbl-mediated negative regulation of cytoplasmic targets.

Fig. 1. Comparable kinase activity of Syk associated with wild-type Cbl and Cbl-70Z mutant. 293T cells were transfected with plasmids encoding wild-type (WT) HA-Cbl, HA-Cbl-70Z (1 µg) or Syk (0.15 µg), as indicated, together with a CD8-ζ (0.5 µg) expression plasmid. The cells were lysed 48 h post-transfection and HA-Cbl proteins were immunoprecipitated (IP) from 1 mg of lysate using the anti-HA antibody. Following washing, half of the immunoprecipitates was used for in vitro kinase assays (A) and the other half was used for anti-HA and anti-Syk immunoblotting (B). The kinase activity was assayed by measuring the incorporation of 32P signal of [γ-32P]ATP into a synthetic substrate (Raytide™). Results in (A) are expressed as the mean ± 1 SD of two or three replicates. Syk protein levels (B, bottom panel) were quantified by densitometry and used to normalize Syk kinase activities in (A). The normalized kinase activity associated with wild-type Cbl versus 70Z is shown in (C). The results shown are representative of three experiments.

Fig. 2. Cbl-dependent ubiquitylation of Syk in 293T cells and the requirement for an intact Cbl RING finger domain. 293T cells were transfected with 0.15 µg of Syk (+) or vector control (–) together with 3 µg of GFP–Cbl (+) or vector control (–). Each transfection also included plasmids encoding HA-ubiquitin (7 µg) and CD8-ζ (0.5 μg). In (A), the cells were treated with 50 µM MG132 (+) or DMSO control (–) for 5 h prior to harvesting the cells. Anti-Syk IPs from 750 µg aliquots of lysate protein were resolved by SDS–PAGE and immunoblotted with anti-HA antibody (top panel) followed by anti-Syk (second panel), and anti-Syk immunoprecipitates from 1.5 mg aliquots of lysate protein were immunoblotted with anti-Syk (third panel). In (B and C), the cells were transfected with 3 µg of wild-type GFP–Cbl (WT), RING finger mutant C3AHN or vector control (–). In (B), anti-Syk IPs from 750 µg aliquots of lysate protein were resolved by SDS–PAGE and immunoblotted with anti-HA antibody (top panel) followed by anti-Syk antibody (middle panel). Aliquots of cell lysates (25 µg) were immunoblotted directly with an anti-GFP antibody to visualize GFP–Cbl (bottom panel). GFP alone, which runs at a lower molecular weight, is not shown. In (C), anti-ζ IPs from 1.5 mg aliquots of lysate protein were immunoblotted with anti-HA antibody (top panel) followed by anti-ζ antibody (middle panel). Aliquots of cell lysates (25 µg) were immunoblotted directly with an anti-pTyr antibody to visualize phosphorylated CD8-ζ. Size markers (kDa) are indicated on the left.

Fig. 3. The Cbl TKB and RF domains are sufficient for Cbl-dependent ubiquitylation of Syk in 293T cells. 293T cells were transfected with the indicated expression plasmids together with HA-ubiquitin and CD8-ζ plasmids as in Figure 2. Anti-Syk IP and serial anti-HA and anti-Syk immunoblotting, and anti-GFP immunoblotting of whole-cell lysates were also as in Figure 2. The transfected GFP–Cbl construct designations are: GFP-full length Cbl (WT) and its G306E mutants (G306E); GFP–Cbl amino acids 1–436 (1–436) and its corresponding G306E mutant (1–436-G306E).

Fig. 6. BCR-induced Syk ubiquitylation in Ramos-T cells and its enhancement by MG132 treatment. The SV40 T-antigen-expressing Ramos B-cell line (Ramos-T) was transiently transfected by electroporation with 10 µg of a plasmid encoding HA-ubiquitin. At 48 h post-transfection, cells were washed and stimulated as in Figure 5 for the indicated times (A) or for 10 min (B). Anti-Syk IPs from 1 mg aliquots of lysate protein were subjected to serial immunoblotting with anti-HA followed by anti-Syk antibody. In (B), cells were treated with 50 µM MG132 (+) or DMSO control (–) 5 h prior to stimulation.

Fig. 8. Inhibition of Syk-dependent NFAT-luciferase activity by Cbl and the requirement for the TKB and RING finger domains. Jurkat cells, p116-T, were transfected with plasmids encoding the NFAT-luciferase reporter (10 µg) and the indicated plasmids encoding Syk (5 µg) or various Cbl proteins (15 µg). The HA-Cbl constructs used were: wild-type (WT), its TKB domain mutant (G306E), RING finger mutants 70Z-Cbl (70Z) and C3AHN (C3AHN), and a truncation construct encoding amino acids 1–436 (1–436). Transfections were performed in duplicate and cells were analyzed in replicates of five. Cells were either left unstimulated or stimulated with PMA plus ionomycin. Mean luciferase activity was expressed as a percentage of values obtained with PMA plus ionomycin treatment. The combined standard error of the mean is displayed. Data are representative of three independent experiments. Aliquots of cell lysates (20 µg) were subjected to serial anti-HA (middle panel) and anti-Syk immunoblotting.